Otitis media (OM), or middle ear infection, is a highly prevalent pediatric disease worldwide, ranking first in reasons why children make physician's office and emergency room visits, undergo surgery requiring general anesthesia, and experience hearing loss. The widely held practice of prescribing of broad-spectrum antibiotics to treat OM has been a major driving force behind the sobering emergence of multiple antibiotic resistant strains of bacteria among those that cause OM. Since the recent licensure of a 7-valent vaccine directed against Streptococcus pneumoniae, the relative proportion of cases of OM due to our studied microorganism (nontypeable Haemophilus influenzae or NTHI) has increased significantly. NTHI are now credited with approximately 40% of all cases of acute OM, yet remain the predominant causative agent of chronic OM, recurrent OM, and OM in children who fail treatment. There is thus a pressing need to develop better methods to manage OM, preferably via the development of vaccines to prevent middle ear infections due to NTHI. We have designed and extensively tested vaccine candidates for the prevention of NTHI-induced OM that have been derived from primarily two bacterial adhesins/virulence determinants: the OMP P5-homologous adhesin (or OMP P5) and the type IV twitching pilus (or Tfp). These candidates have shown significant promise to date in pre-clinical efficacy trials, however, there are as yet a few gaps in the understanding required to complete their development. Thereby, herein, we propose two complementary specific aims to gain this needed enhanced understanding, as well as a third integrated specific aim designed to translate the success realized to date using traditional immunization approaches for these vaccine candidates to methods of delivery that are novel and non-invasive, and thereby have a significant opportunity to improve the health of children worldwide. In Specific Aim 1, we will determine niche-specific and concordant vs. discordant expression of our targeted virulence determinants, as well as define the cognate receptor(s) for these ligands and determine their relative spatial distribution in the uppermost airway. In Specific Aim 2, we will determine the biological mechanisms by which these candidate antigens have induced significant protection against experimental NTHI-induced OM when delivered parenterally (e.g. the correlates of immune protection). In Specific Aim 3, we will develop and assess three novel non-invasive delivery methods (intranasal, transcutaneous and sublingual) for these promising vaccine candidates and determine their relative protective efficacy when delivered via each of these routes.